Title : Non - nociceptive afferent activity depresses nocifensive 1 behavior and nociceptive synapses via an endocannabinoid -

46 Previously, low frequency stimulation (LFS) of a non-nociceptive touch-sensitive neuron 47 has been found to elicit endocannabinoids-dependent long-term depression (eCB-LTD) 48 in nociceptive synapses in the leech central nervous system (CNS) that requires 49 activation of a presynaptic TRPV-like receptor by postsynaptically-synthesized 2-AG. 50 This capacity of non-nociceptive afferent activity to reduce nociceptive signaling 51 resembles gate control of pain, albeit longer lasting in these synaptic experiments. 52 Having observed eCB-LTD at a single sensory-motor synapse, this study examines the 53 functional relevance of this mechanism. Specifically, whether this form of synaptic 54 plasticity has similar effects at the behavioral level in which additional, intersegmental 55 neural circuits are engaged. Experiments were carried out using a semi-intact 56 preparation that permitted both synaptic recordings and monitoring of the leech whole57 body shortening, a defensive withdrawal reflex that was elicited via intracellular 58 stimulation of a single nociceptive neuron (the N-cell). The same LFS of a non59 nociceptive afferent that induced eCB-LTD in single synapses also produced an 60 attenuation of the shortening reflex. Similar attenuation of behavior was also observed 61 when 2-AG was applied. LFS-induced behavioral and synaptic depression was blocked 62 by tetrahydrolipstatin (THL), a DAG lipase inhibitor, and by SB366791, a TRPV1 63 antagonist. The effects of both THL and SB366791 were observed following either bath64 application of the drug or intracellular injection into the presynaptic (SB366791) or 65 postsynaptic (THL) neuron. These findings demonstrate a novel, endocannabinoid66 based mechanism by which non-nociceptive afferent activity may modulate nocifensive 67 behaviors via action on primary afferent synapses. 68